Isoquinolinyl triazolone complexes

ABSTRACT

A complex of a compound of Formula 1, 
     
       
         
         
             
             
         
       
     
     a stereoisomer thereof, or a tautomer of the compound of Formula 1 or stereoisomer thereof, and a cyclodextrin, in which the complex is an amorphous solid. This disclosure also relates to materials and methods for preparing the complex, to pharmaceutical compositions which contain the complex, and to the use of the complex to treat Type I hypersensitivity reactions, autoimmune diseases, inflammatory disorders, cancer, non-malignant proliferative disorders, and other conditions associated with BTK.

FIELD OF THE INVENTION

This invention relates to isoquinolinyl triazolone complexes which areinhibitors of Bruton's tyrosine kinase (BTK) and to materials andmethods used to prepare the complexes. This invention also relates topharmaceutical compositions which contain the isoquinolinyl triazolonecomplexes, and to the use of the complexes to treat diseases, disorders,and conditions associated with BTK.

BACKGROUND OF THE INVENTION

BTK is a member of the TEC family of non-receptor protein tyrosinekinases, and it is involved in the regulation of B-cell development,activation, and survival through B-cell antigen receptor (BCR)signaling. See W. N. Khan et al., Immunity 3:283-299 (1995); and A. B.Satterthwaite and O. N. Witte, Immunol. Rev. 175:120-127 (2000).Mutation of the gene encoding BTK in humans leads to a condition knownas X-linked agammaglobulinemia (XLA), which is characterized by reducedimmune function, including impaired maturation of B cells, decreasedlevels of immunoglobulin and peripheral B cells, diminished T-cellindependent immune response, and attenuated calcium mobilizationfollowing BCR stimulation. See F. S. Rosen et al., N. Engl. J. Med.333(7):431-440 (1995); and J. M. Lindvall et al., Immunol. Rev.203:200-215 (2005).

BTK's key role in B-cell development and the BCR signaling pathwaysuggests that inhibition of BTK may provide therapeutic benefit for thetreatment of lymphoma, inflammatory disorders, and autoimmune diseases,among others. Clinical studies involving the depletion of mature B cellsvia treatment with rituximab indicate that rheumatoid arthritis,systemic lupus erythematosus (SLE), and multiple sclerosis may resultfrom the over expression of B cells. See J. C. Edwards et al., N. Engl.J Med. 350:2572-81 (2004); C. Favas and D. A. Isenberg Nat. Rev.Rheumatol. 5:711-16 (2009); and S. L. Hauser et al. N. Engl. J. Med.358:676-88 (2008). Other studies suggest that the BCR pathway may beinvolved in the survival of tumor cells in non-Hodgkin lymphoma anddiffuse large B-cell lymphoma. See R. Küeppers, Nat. Rev. Cancer5:251-62 (2005); and R. E. Davis et al., Nature 463:88-92 (2010). Inpreclinical studies, BTK-deficient mice have demonstrated decreaseddisease progression in murine models of SLE and resistance tocollagen-induced arthritis. See M. J. Shlomchik et al., J. Exp. Med.180:1295-1306 (1994); and L. Jansson and R. Holmdahl, Clin. Exp.Immunol. 94(3):459-65 (1993). Furthermore, a selective irreversible BTKinhibitor has been shown to completely suppress collagen-inducedarthritis in mice, to inhibit autoantibody production and thedevelopment of kidney disease in a mouse model for SLE, and to induceobjective clinical responses in dogs with spontaneous B-cell non-Hodgkinlymphoma. See L. A. Honigberg et al., Proc. Natl. Acad. Sci. USA107(29):13075-80 (2010).

Certain inhibitors of Bruton's tyrosine kinase are described in WO99/54286 A2, WO 2002/50071 A1, WO 2007/087068 A2, WO 2008/039218 A2, WO2008/121742 A2, WO 2007/147771 A2, WO 2009/077334 A1, WO 2009/098144 A1,WO 2009/156284 A1, WO 2010/000633 A1, WO 2010/006947 A1, WO 2008/033834A1, WO 2010/056875 A1, WO 2010/068788 A1, and WO 2010/068810 A2.

Published international application WO 2014/164558 A1 (the '558application) describes the preparation, characterization, and use ofvarious pyridinyl and fused pyridinyl triazolones which are inhibitorsof BTK. Included among the compounds in the '558 application is theisoquinolinyl triazolone,(S)-3-(1-((1-acryloylpyrrolidin-3-yl)oxy)isoquinolin-3-yl)-1H-1,2,4-triazol-5(4H)-one.See Example 5 of the '558 application. Though a potent inhibitor of BTK,the crystalline compound prepared in Example 5 in the '558 applicationhas low aqueous solubility, which may limit its adsorption andbioavailability following oral dosing.

SUMMARY OF THE INVENTION

This invention provides solid complexes of isoquinolinyl triazolones andcyclodextrin. The complexes are amorphous solids which exhibit improvedaqueous solubility and bioavailability over the correspondingcrystalline forms of the compounds. This invention also providesmaterials and methods for preparing the complexes, pharmaceuticalcompositions which contain the complexes, and the use of the complexesto treat diseases, disorders, and conditions associated with BTK.

One aspect of the invention provides a complex comprising a compound ofFormula 1,

a stereoisomer thereof or a tautomer of the compound of Formula 1 orstereoisomer thereof,

and a cyclodextrin, wherein the complex is an amorphous solid.

Another aspect of the invention provides a pharmaceutical compositionwhich includes a complex as defined above; and a pharmaceuticallyacceptable excipient.

A further aspect of the invention provides a method of making a complexas defined above, the method comprising atomizing a liquid solution intodroplets, the liquid solution comprising a compound, stereoisomer ortautomer as defined above, a cyclodextrin derivative, and water, andremoving at least a portion of the water from the droplets to form thecomplex.

An additional aspect of the invention provides a complex as definedabove for use as a medicament.

Another aspect of the invention provides a complex as defined above, forthe manufacture of a medicament for the treatment of a conditionassociated with BTK.

A further aspect of the invention provides a method for inhibiting BTKin a subject, the method comprising administering to the subject acomplex as defined above.

An additional aspect of the invention provides a method of treating adisease, disorder or condition associated with BTK in a subject, themethod comprising administering to the subject an effective amount of acomplex as defined above.

Another aspect of the invention provides a method of treating a disease,disorder or condition in a subject, the method comprising administeringto the subject an effective amount of a complex as defined above,wherein the disease, disorder or condition is selected from Type Ihypersensitivity reactions, autoimmune diseases, inflammatory disorders,cancer, and non-malignant proliferative disorders.

A further aspect of the invention provides a method of treating adisease, disorder or condition in a subject, the method comprisingadministering to the subject an effective amount of a complex as definedabove, wherein the disease, disorder or condition is selected fromallergic rhinitis, asthma, atopic dermatitis, rheumatoid arthritis,multiple sclerosis, systemic lupus erythematosus, lupus nephritis,psoriasis, immune thrombocytopenic purpura, inflammatory bowel disease,chronic obstructive pulmonary disease, Sjögren's syndrome, ankylosingspondylitis, Behcet's disease, pemphigus vulgaris, idiopathicplasmacytic lymphadenopathy, atherosclerosis, myocardial infarction, andthrombosis.

An additional aspect of the invention provides a method of treating adisease, disorder or condition in a subject, the method comprisingadministering to the subject an effective amount of a complex as definedabove, wherein the disease, disorder or condition is selected fromB-cell lymphoma, chronic lymphocytic leukemia, and multiple myeloma.

Another aspect of the invention provides a combination of an effectiveamount of a complex as defined above, and at least one additionalpharmacologically active agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the mean concentration of the compound of Example 1 inblood as a function of time following oral dosing of dogs withFormulations A, B, C, D, E, and F.

FIG. 2 shows the mean eluent concentration of the compound of Example 1as a function of time for Formulations A, B, C, and E, which wereevaluated in the Japanese Pharmacopoeia Dissolution Test.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise indicated, this disclosure uses definitions providedbelow.

“About” or “approximately,” when used in connection with a measurablenumerical variable, refers to the indicated value of the variable and toall values of the variable that are within the experimental error of theindicated value or within +10 percent of the indicated value, whicheveris greater.

“Condition associated with BTK” and similar phrases relate to a disease,disorder or condition in a subject for which inhibition of BTK mayprovide a therapeutic or prophylactic benefit.

“Drug,” “drug substance,” “active pharmaceutical ingredient,” and thelike, refer to a compound (e.g., compound of Formula 1) that may be usedfor treating a subject in need of treatment.

“Drug product,” “pharmaceutical dosage form,” “dosage form,” “finaldosage form” and the like, refer to a pharmaceutical compositionsuitable for treating a subject in need of treatment and generally maybe in the form of tablets, capsules, sachets containing powder orgranules, patches, films, and the like.

“Effective amount” of a drug, “therapeutically effective amount” of adrug, and the like, refer to the quantity of the drug that may be usedfor treating a subject and may depend on the weight and age of thesubject and the route of administration, among other things.

“Excipient” refers to any diluent or vehicle for a drug.

“Opposite enantiomer” refers to a molecule that is a non-superimposablemirror image of a reference molecule, which may be obtained by invertingall of the stereogenic centers of the reference molecule. For example,if the reference molecule has S absolute stereochemical configuration,then the opposite enantiomer has R absolute stereochemicalconfiguration. Likewise, if the reference molecule has S,S absolutestereochemical configuration, then the opposite enantiomer has R,Rstereochemical configuration, and so on.

“Pharmaceutical composition” refers to the combination of one or moredrug substances and one or more excipients.

“Pharmaceutically acceptable” substances refer to those substances whichare suitable for administration to subjects.

“Pure stereoisomer” and variants thereof refer to a sample containing acompound which has a specific stereochemical configuration and whichcomprises at least about 99.5% of the sample.

“Stereoisomer” and “stereoisomers” of a compound with givenstereochemical configuration refer to the opposite enantiomer of thecompound and to any diastereoisomers, including geometrical isomers(ZIE) of the compound. For example, if a compound has S,R,Zstereochemical configuration, its stereoisomers would include itsopposite enantiomer having R,S,Z configuration, and its diastereomershaving S,S,Z configuration, R,R,Z configuration, S,R,E configuration,R,S,E configuration, S,S,E configuration, and R,R,E configuration. Ifthe stereochemical configuration of a compound is not specified, then“stereoisomer” refers to any one of the possible stereochemicalconfigurations of the compound.

“Substantially pure stereoisomer” and variants thereof refer to a samplecontaining a compound having a specific stereochemical configuration andwhich comprises at least about 95% of the sample.

“Subject” refers to a mammal, including a human.

“Substituted,” when used in connection with a chemical substituent ormoiety (e.g., a C₁₋₆ alkyl group), means that one or more hydrogen atomsof the substituent or moiety have been replaced with one or morenon-hydrogen atoms or groups, provided that valence requirements are metand that a chemically stable compound results from the substitution.

“Treating” refers to reversing, alleviating, inhibiting the progress of,or preventing a disease, disorder or condition to which such termapplies, or to reversing, alleviating, inhibiting the progress of, orpreventing one or more symptoms of such disorder, disease or condition.

“Treatment” refers to the act of “treating,” as defined immediatelyabove.

The following abbreviations may be used in the specification: Ac(acetyl); ACN (acetonitrile); AIBN (azo-bis-isobutyronitrile); API(active pharmaceutical ingredient); aq (aqueous); Boc(tert-butoxycarbonyl); Cbz (carbobenzyloxy); CDI(1,1′-carbonyldiimidazole); dba (dibenzylideneacetone); DBU(1,8-diazabicyclo[5.4.0]undec-1(7)-ene); DCC(1,3-dicyclohexylcarbodiimide); DCM (dichloromethane); DIPEA(N,N-diisopropylethylamine, Hünig's Base); DMA (N,N-dimethylacetamide);DMAP (4-dimethylaminopyridine); DMARD (disease modifying antirheumaticdrug); DME (1,2-dimethoxyethane); DMF (N,N-dimethylformamide); DMSO(dimethylsulfoxide); DPPA (diphenylphosphoryl azide); dppf(1,1′-bis(diphenylphosphino)ferrocene); DTT (dithiothreitol); EDAethoxylated dodecyl alcohol, Brj® 35); EDC(N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide); EDTA(ethylenediaminetetraacetic acid); ee (enantiomeric excess); eq(equivalents); Et (ethyl); Et₃N (triethyl-amine); EtOAc (ethyl acetate);EtOH (ethanol); 5-FAM (5-carboxyfluorescein); HATU(2-(3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate(V)); HEPES(4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid); HOAc (aceticacid); HOBt (1H-benzo[d][1,2,3]triazol-1-ol); IC₅₀ (concentration at 50%inhibition); IPA (isopropanol); IPAc (isopropyl acetate); IPE(isopropylether); LDA (lithium diisopropylamide); LiHMDS (lithiumbis(trimethylsilyl)amide); mCPBA (m-chloroperoxybenzoic acid); Me(methyl); MeOH (methanol); MTBE (methyl tert-butyl ether); mp (meltingpoint); NaOt-Bu (sodium tertiary butoxide); NMM (N-methylmorpholine);NMP (1-methyl-2-pyrrolidinone); PE (petroleum ether); Ph (phenyl); pIC₅₀(−log₁₀(IC₅₀), where IC₅₀ is given in molar (M) units); Pr (propyl);i-Pr (isopropyl); PTFE (polytetrafluoroethylene); RT (room temperature,approximately 20° C. to 25° C.); TCEP (tris(2-carboxyethyl)phosphine);Tf (trifluoromethylsulfonyl); TFA (trifluoroacetic acid); TFAA(2,2,2-trifluoroacetic anhydride); THF (tetrahydrofuran); TMS(trimethylsilyl); and Tris buffer(2-amino-2-hydroxymethyl-propane-1,3-diol buffer).

This disclosure describes a complex of a compound of Formula 1, astereoisomer thereof, or a tautomer of the compound of Formula 1 orstereoisomer thereof, and a cyclodextrin. The complex is an amorphoussolid which exhibits improved aqueous solubility and bioavailabilityover the corresponding crystalline form of the compound. This disclosurealso concerns materials and methods for preparing the complex,pharmaceutical compositions which contain the complex, and the use ofthe complex to treat diseases, disorders, and conditions associated withBTK, including Type I hypersensitivity reactions, autoimmune diseases,inflammatory disorders, cancer, non-malignant proliferative disorders,and other diseases, disorders or conditions associated with BTK.

As used in this disclosure, “cyclodextrin” refers to cyclicoligosaccharides consisting of (α-1,4)-linked α-D-glucopyranose units.Each subunit of a naturally-occurring (unmodified or parent)cyclodextrin has secondary hydroxy groups at the 2- and 3-positions anda primary hydroxy group at the 6-position. A cyclodextrin may be thoughtof as a toroid or hollow truncated cone, which because of the locationof the hydroxy groups has a hydrophilic exterior surface and acomparatively less lipophilic internal cavity. The internal cavity maycapture at least a portion of a drug molecule, such as the compound ofFormula 1, which results in the formation of an inclusion complex.Covalent bonds are neither made nor broken during the formation of thedrug-cyclodextrin complex. In aqueous solution, the complex dissociates,resulting in free drug molecules in equilibrium with drug moleculesbound in the cyclodextrin cavities. Unless stated otherwise,cyclodextrin refers to both unmodified cyclodextrins and tochemically-modified cyclodextrins, i.e., a “cyclodextrin derivative.”

“Cyclodextrin derivative” refers to a structural analog of a parentcyclodextrin in which one or more of the 2-hydroxy, 3-hydroxy, and6-hydroxy groups in the α-D-glucopyranose subunits are chemicallymodified,

For naturally-occurring cyclodextrins, each R in Formula A is hydrogen,whereas for cyclodextrin derivatives, at least one R is non-H. For bothnaturally-occurring and chemically modified cyclodextrins,α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin correspond to n being6, 7, and 8 in Formula A, respectively.

Table 1 lists exemplary cyclodextrins and corresponding values of R inFormula A. Exemplary cyclodextrins include naturally-occurring α-, β-,and γ-cyclodextrins, as well as α-, β-, and γ-cyclodextrin derivativessuch as (2-hydroxypropyl)-β-cyclodextrin, randomly methylatedβ-cyclodextrin, sulfobutylether β-cyclodextrin, and(2-hydroxypropyl)-γ-cyclodextrin. Other useful cyclodextrin derivativesinclude methyl-, dimethyl-, and trimethyl-β-cyclodextrin(tri-O-methyl-β-cyclodextrin); randomly dimethylated-β-cyclodextrin;ethyl-, diethyl-, and triethyl-β-cyclodextrin(tri-O-ethyl-β-cyclodextrin); (2-hydroxyethyl)-β-cyclodextrin and(β-hydroxypropyl)-β-cyclodextrin; (2,3-dihydroxypropyl)-β-cyclodextrinand (2-hydroxyisobutyl)-β-cyclodextrin; carboxymethyl-β-cyclodextrin andcarboxymethyl ethyl-β-cyclodextrin; tributyryl-β-cyclodextrin(tri-O-butyryl-β-cyclodextrin), trivaleryl-β-cyclodextrin(tri-O-valeryl-β-cyclodextrin), and dihexanoyl-β-cyclodextrin(di-O-hexanoyl-β-cyclodextrin); glucosyl-β-cyclodextrin(6-O-α-D-glucosyl-β-cyclodextrin) and maltosyl-β-cyclodextrin(6-O-α-maltosyl-β-cyclodextrin). For a discussion of cyclodextrin indrug products, see M. E. Davis and M. E. Brewster, Nat. Rev. Drug Disc.3(12):1023-1035 (2004); see also R. C. et al., AAPS PharmSciTech6(2):329-357 (2005).

TABLE 1 Cyclodextrins Name R in Formula A α-cyclodextrin (unmodified) Hβ-cyclodextrin (unmodified) H γ-cyclodextrin (unmodified) Hmethyl-β-cyclodextrin —CH₃ or —H dimethyl-β-cyclodextrin —CH₃ or —Htrimethyl-β-cyclodextrin —CH₃ randomly methylated-β-cyclodextrin —CH₃ or—H randomly dimethylated-β-cyclodextrin —CH₃ or —H ethyl-β-cyclodextrin—CH₂CH₃ or —H diethyl-β-cyclodextrin —CH₂CH₃ or —Htriethyl-β-cyclodextrin —CH₂CH₃ (2-hydroxyethyl)-β-cyclodextrin—CH₂CH₂OH or —H (2-hydroxypropyl)-β-cyclodextrin —CH₂CHOHCH₃ or —H(2-hydroxypropyl)-γ-cyclodextrin —CH₂CHOHCH₃ or —H(3-hydroxypropyl)-β-cyclodextrin —(CH₂)₃OH or —H(2,3-dihydroxypropyl)-β-cyclodextrin —CH₂CHOHCH₂OH or —H(2-hydroxyisobutyl)-β-cyclodextrin —CH₂C(CH₃)₂OH or —Hcarboxymethyl-β-cyclodextrin —CH₂C(O)O⁻M⁺ or —H carboxymethylethyl-β-cyclodextrin —CH₂C(O)O⁻M⁺, —CH₂CH₃ or —Htributyryl-β-cyclodextrin —C(O)(CH₂)₂CH₃ trivaleryl-β-cyclodextrin—C(O)(CH₂)₃CH₃ dihexanoyl-β-cyclodextrin —C(O)(CH₂)₄CH₃ or —Hsulfobutylether β-cyclodextrin —(CH₂)₄SO₃ ⁻M⁺ or —Hglucosyl-β-cyclodextrin -glucosyl or —H maltosyl-β-cyclodextrin-maltosyl or —H

In Table 1, M⁺ represents a pharmaceutically acceptable cationicspecies, which includes H⁺, Li⁺, Na⁺, K⁺, and NH₄ ⁺, among others. Thus,for example, carboxymethyl-β-cyclodextrin and carboxymethylethyl-β-cyclodextrin may include at least one R which is —CH₂C(O)O⁻Na⁺,and sulfobutylether β-cyclodextrin may include at least one R which is—(CH₂)₄SO₃Na⁺.

The compound of Formula 1, a stereoisomer thereof, or a tautomer of thecompound of Formula 1 or stereoisomer thereof, may form inclusioncomplexes with any one of the cyclodextrins listed in Table 1,including, for example, naturally-occurring β-cyclodextrin andγ-cyclodextrin, chemically modified β-cyclodextrin derivatives(2-hydroxypropyl)-β-cyclodextrin, methyl β-cyclodextrin, andsulfobutylether β-cyclodextrin, and chemically modified γ-cyclodextrinderivative (2-hydroxypropyl)-γ-cyclodextrin. Naturally-occurring(unmodified) β-cyclodextrin and γ-cyclodextrin are commerciallyavailable from Wacker Chemie AG under the trade names CAVAMAX® W7 PHARMAand CAVAMAX® W8 PHARMA. Likewise chemically modified cyclodextrinderivatives (2-hydroxypropyl)-β-cyclodextrin,(2-hydroxypropyl)-γ-cyclodextrin, and methyl β-cyclodextrin areavailable under the trade names CAVASOL® W7 HP PHARMA, CAVASOL® W8 HPPHARMA, and CAVASOL® W7 M PHARMA, respectively. CAVASOL® W7 HP PHARMAhas a chemical structure according to Formula A in which n is 7 and R is(—CH₂CHOHCH₃)_(t) or (—H)_(21-t) and t is about 4.1 to about 5.1;CAVASOL® W8 HP PHARMA has a chemical structure according to Formula A inwhich n is 8 and R is (—CH₂CHOHCH₃)_(t) or (—H)_(24-t) and t is about 4to about 5.6; and CAVASOL® W7 M PHARMA has a chemical structure in whichn is 7 and R is (—CH₃)_(t) or (—H)₂₁-t and t is about 11 to about 14.Useful sulfobutylether β-cyclodextrins are commercially available fromCyDex Pharmaceuticals, Inc. under the trade name CAPTISOL®, which has achemical structure according to Formula A in which n is 7 and R is(—(CH₂)₄SO₃—Na⁺)_(t) or (—H)₂₁-t and t is about 6 to about 7.1.

The complex is an amorphous solid. The term “amorphous” refers to astate in which the material lacks long range order at the molecularlevel and, depending upon temperature, may exhibit the physicalproperties of a solid or a liquid. Typically such materials do not givedistinctive X-ray diffraction patterns and, while exhibiting theproperties of a solid, are more formally described as a liquid. Uponheating, a change from solid to liquid properties occurs which ischaracterized by a change of state, typically second order (“glasstransition”). The term “crystalline” refers to a solid phase in whichthe material has a regular ordered internal structure at the molecularlevel and gives a distinctive X-ray diffraction pattern with definedpeaks. Such materials when heated sufficiently will also exhibit theproperties of a liquid, but the change from solid to liquid ischaracterized by a phase change, typically first order (“meltingpoint”).

The compound of Formula 1 may exist as polymorphs, stereoisomers,tautomers, or some combination thereof, and may be isotopically-labeled.

The compound of Formula 1 may exist as stereoisomer(S)-3-(1-((1-acryloylpyrrolidin-3-yl)oxy)isoquinolin-3-yl)-1H-1,2,4-triazol-5(4H)-one,

or as stereoisomer(R)-3-(1-((1-acryloylpyrrolidin-3-yl)oxy)isoquinolin-3-yl)-1H-1,2,4-triazol-5(4H)-one,

The stereoisomers (i.e., the enantiomer and its opposite enantiomer) maybe pure, substantially pure, or a mixture.

The compound of Formula 1 or stereoisomer thereof may exist astautomers, which are isomers resulting from tautomerization, includingimine-enamine, keto-enol, oxime-nitroso, and amide-imidic acidtautomerism. The triazolone moiety of Formula 1 may exist, for example,in the following tautomeric forms:

The compound of Formula 1 may exhibit more than one type of isomerism.

The compound of Formula 1, stereoisomer thereof, or tautomer of thecompound of Formula 1 or stereoisomer thereof, may possess isotopicvariations, in which at least one atom is replaced by an atom having thesame atomic number, but an atomic mass different from the atomic massusually found in nature. Isotopes suitable for inclusion in compounds ofFormula 1 include, for example, isotopes of hydrogen, such as ²H and ³H;isotopes of carbon, such as ¹¹C, ¹³C and ¹⁴C; isotopes of nitrogen, suchas ¹³N and ¹⁵N; and isotopes of oxygen, such as ¹⁵O, ¹⁷O and ¹⁸O. Use ofisotopic variations (e.g., deuterium, ²H) may afford certain therapeuticadvantages resulting from greater metabolic stability, for example,increased in vivo half-life or reduced dosage requirements.Additionally, certain isotopic variations of the disclosed compounds mayincorporate a radioactive isotope (e.g., tritium, ³H, or ¹⁴C), which maybe useful in drug and/or substrate tissue distribution studies.Substitution with positron emitting isotopes, such as ¹¹C, ¹⁵O and ¹³N,may be useful in Positron Emission Topography (PET) studies forexamining substrate receptor occupancy. Isotopically-labeled compoundsmay be prepared by processes analogous to those described elsewhere inthe disclosure using an appropriate isotopically-labeled reagent inplace of a non-labeled reagent.

The complex may be prepared from the compound of Formula 1 or from astereoisomer thereof, or from a tautomer of the compound of Formula 1 orstereoisomer thereof, by spray drying, lyophilization, and othermethods. Spray drying involves dissolving the compound and cyclodextrinin one or more compatible solvents, atomizing the resulting solution,and evaporating the solvent or solvents to form the complex.Lyophilization or freeze drying also involves dissolving the compoundand cyclodextrin in a compatible solvent (usually water), rapidlyfreezing the solution, and removing the solvent via sublimation(typically under vacuum) and desorption. For more detailed descriptionof lyophilization, see Georg-Wilhelm Oetjen, “Freeze-Drying,” Ullmann'sEncyclopedia of Industrial Chemistry (2004).

For each of these methods, the fraction of drug substance that isamorphous is in the range of about 50% w/w to about 100% w/w, from about75% w/w to about 100% w/w, from about 90% w/w to about 100% w/w, or fromabout 95% w/w to about 100% w/w, based on the total mass of the compoundof Formula 1. Ideally, the fraction of drug that is amorphous is in therange of about 99% w/w to about 100% w/w, based on the total mass of thecompound of Formula 1.

As noted above, the complex may be prepared by spray drying, whichincludes dissolving the compound of Formula 1 in one or more compatiblesolvents to form a solution. Generally, a compatible solvent is anyliquid which will dissolve the compound of Formula 1 and thecyclodextrin. In practice, a compatible solvent includes any liquidwhich at room temperature will completely dissolve the compound ofFormula 1 and the cyclodextrin at respective concentrations of about0.5% w/w or greater, about 1% w/w or greater, or more typically, atconcentrations of about 5% w/w or greater. Useful solvents include thosewhich are volatile, have a normal boiling point of about 150° C. orless, exhibit relatively low toxicity, and can be removed from theresulting complex such that the level of solvent in the drug productmeets the International Committee on Harmonization (ICH) guidelines forresidual solvent. Additional processing, such as tray-drying, may berequired to meet ICH residual solvent levels.

The complex is advantageously prepared using water as the solvent.Although the compound of Formula 1 is poorly soluble in water at neutralpH and below, its aqueous solubility increases with increasing basicity,so adjusting the pH of water to about 10 or above, to about 11 or above,to about 12 or above, or to about 13 or above, improves aqueoussolubility. Thus, the compound of Formula 1 may be first dissolved in anaqueous base, such as NaOH, KOH, and the like, and then mixed with anaqueous cyclodextrin solution. The concentration of the base may rangefrom about 10⁻⁴M to about 1M, from about 10⁻³M to about 1M, from about10⁻²M to about 1M, or from about 10⁻¹M to about 1N. The solution may beprepared by adding the compound of Formula 1 to the aqueous base withconcurrent or subsequent mixing, and then adding cyclodextrin withconcurrent or subsequent mixing. The cyclodextrin is typically added asan aqueous solution with a concentration of about 1-60% w/v, about10-50% w/v, about 20-40% w/v, or about 40% w/v). Mixing may be carriedout using mechanical means, e.g., through the use of overhead mixers,magnetically driven mixers or stirring bars, planetary mixers, orhomogenizers. The pH of the resulting solution may be adjusted to a pHof about 7 and the aqueous solution spray-dried (e.g., lyophilized).

The compound of Formula 1 and cyclodextrin may be added to the aqueousbase up to their respective solubility limits, but to ensure completedissolution, the amount added is usually less than about 80% of thesolubility limit at the solution temperature. The concentration of thecompound of Formula 1 typically ranges from about 0.1% w/w to about 10%w/w depending on its solubility, and the concentration of cyclodextrintypically ranges from about 0.1% w/w to about 20% w/w. The concentrationof the compound of Formula 1 in the solution is typically at least about0.1%, 0.5%, 1%, or 5% w/w, and the amount of cyclodextrin is typicallyabout 1 to about 20 times the amount of active compound based on molarconcentration. Although increasing the concentration of the activecompound and cyclodextrin reduces the volume of solvent, higherconcentrations of the active agent and cyclodextrin may be too viscousto atomize efficiently into small droplets. A solution viscosity ofabout 0.5 cp to about 50,000 cp or about 10 cp to about 2,000 cpgenerally results in satisfactory atomization.

The solution comprising the compound of Formula 1, cyclodextrin, andsolvent (usually water) is delivered to an atomizer that breaks thesolution into small droplets. Useful atomizers include “pressure” orsingle-fluid nozzles; two-fluid nozzles; centrifugal or spinning-diskatomizers; ultrasonic nozzles; and mechanical vibrating nozzles.Detailed descriptions of atomization processes can be found in Lefebvre,Atomization and Sprays (1989), and in Perry's Chemical Engineers'Handbook (7th ed. 1997). Generally, the droplets produced by theatomizer are less than about 500 μm in diameter when they exit theatomizer.

Once atomized, at least a portion of the solvent (e.g., water) isremoved from the solution to produce a plurality of solid particlescomprising the complex. The amount of solvent removed to form solidparticles depends on the solubility of the complex in the solvent andthe concentration of complex in the solution prior to atomization.Generally at least about 60% w/w of the solvent originally present inthe solution is removed to form solid particles. The greater the amountof solvent removed from the solution, the less likely crystallinecompound is formed. Thus, the amount of solvent removed from thesolution to form the amorphous complex is typically at least 70% w/w, atleast 80% w/w, or at least 90% w/w.

Atomization and solvent removal occur in a chamber where processconditions may be controlled. The driving force for solvent removal isgenerally provided by maintaining the partial pressure of the solvent inthe chamber below the vapor pressure of the solvent at the temperatureof the drying droplets. This may be accomplished by maintaining apartial vacuum in the chamber (e.g., total pressure of about 0.01atmospheres to about 0.50 atmospheres), by mixing the liquid dropletswith a warm drying gas, or both. Some of the energy required forevaporation of solvent may be provided by heating the solution prior toatomization, though generally the energy comes primarily from the dryinggas. The solution temperature may range from just above the solvent'sfreezing point to about 20° C. or more above its normal boiling point,which is achieved by pressurizing the solution. Solution flow ratesthrough the atomizer may vary depending on the type of nozzle, the sizeof the chamber, and the drying conditions, which include the inlettemperature and the flow rate of the drying gas through the chamber.

The drying gas may, in principle, be essentially any gas, but for safetyreasons and to minimize undesirable oxidation of the complex, theprocess may employ an inert gas such as nitrogen, nitrogen-enriched airor argon. The drying gas is generally introduced into the chamber at atemperature of about 60° C. to about 240° C.

The large surface-to-volume ratio of the droplets and the large drivingforce for evaporation of solvent leads to rapid solidification times forthe droplets. Solidification times of about 20 seconds or less, of about10 seconds or less, or of about 1 second or less are typical. Rapidsolidification helps maintain uniformity and homogeneity of theamorphous complex within and among particles.

The solid particles may remain in the chamber for about 5 seconds toabout 60 seconds following solidification, during which time additionalsolvent evaporates from the particles. Generally, the solvent level ofthe complex as it exits the chamber is less than about 10% w/w and isoften less than 2% w/w. Following formation, the complex may be dried toremove residual solvent using a suitable process, including tray drying,fluid bed drying, microwave drying, belt drying, rotary drying, orvacuum drying. After drying, residual solvent level is typically lessthan about 1% w/w and is often less than about 0.1% w/w.

The resulting spray-dried complex is usually in the form of smallparticles. The mean (volume) diameter of the particles may be less thanabout 1000 m, less than about 500 m, less than about 100 m, less thanabout 50 μm, or less than about 25 μm. The size of the particles may bedetermined by sieve analysis, microscopy, light scattering, orsedimentation. Useful equipment for measuring particle size includesCoulter Counters, Malvern Particle Size Analyzers, and the like. See,e.g., A. R. Gennaro (ed.), Remington: The Science and Practice ofPharmacy (20th ed., 2000).

For a general description of spray-drying processes and spray-dryingequipment, see Perry's Chemical Engineers' Handbook, pages 20-54 to20-57 (6th ed., 1984). Further details of spray drying processes andequipment may be found in Marshall, “Atomization and Spray-Drying,”Chem. Eng. Prog. Monogr. Series 2, 50 (1954); see also, Masters, SprayDrying Handbook (4th ed., 1985) and U.S. Pat. No. 6,763,607.

The complex of the compound of Formula 1, stereoisomer thereof, ortautomer of the compound of Formula 1 or stereoisomer thereof, may beadministered alone or in combination with one or more pharmacologicallyactive compounds. Generally, one or more these compounds areadministered as a pharmaceutical composition (a formulation) inassociation with one or more pharmaceutically acceptable excipients. Thechoice of excipients depends on the particular mode of administration,the effect of the excipient on solubility and stability, and the natureof the dosage form, among other things. Useful pharmaceuticalcompositions and methods for their preparation may be found, forexample, in A. R. Gennaro (ed.), Remington: The Science and Practice ofPharmacy (20th ed., 2000).

The complex may be administered orally. Oral administration may involveswallowing in which case the compound enters the bloodstream via thegastrointestinal tract. Alternatively or additionally, oraladministration may involve mucosal administration (e.g., buccal,sublingual, supralingual administration) such that the compound entersthe bloodstream through the oral mucosa.

Formulations suitable for oral administration include solid andsemi-solid systems such as tablets; soft or hard capsules containingmulti- or nano-particulates or powders; lozenges; chews; gels; fastdispersing dosage forms; films; and buccal or mucoadhesive patches.

The complex may also be used in fast-dissolving, fast-disintegratingdosage forms such as those described in Liang and Chen, Expert Opinionin Therapeutic Patents (2001) 11(6):981-986.

For tablet dosage forms, depending on dose, the active pharmaceuticalingredient (API) may comprise from about 1 wt % to about 80 wt % of thedosage form or more typically from about 5 wt % to about 60 wt % of thedosage form. In addition to the API, tablets may include one or moredisintegrants, binders, diluents, surfactants, glidants, lubricants,anti-oxidants, colorants, flavoring agents, preservatives, andtaste-masking agents. Examples of disintegrants include sodium starchglycolate, sodium carboxymethyl cellulose, calcium carboxymethylcellulose, croscarmellose sodium, crospovidone, polyvinylpyrrolidone,methyl cellulose, microcrystalline cellulose, C₁₋₆ alkyl-substitutedhydroxypropylcellulose, starch, pregelatinized starch, and sodiumalginate. Generally, the disintegrant will comprise from about 1 wt % toabout 25 wt % or from about 5 wt % to about 20 wt % of the dosage form.

Binders are generally used to impart cohesive qualities to a tabletformulation. Suitable binders include microcrystalline cellulose,gelatin, sugars, polyethylene glycol, natural and synthetic gums,polyvinylpyrrolidone, pregelatinized starch, hydroxypropylcellulose andhydroxypropylmethylcellulose. Tablets may also contain diluents, such aslactose (monohydrate, spray-dried monohydrate, anhydrous), mannitol,xylitol, dextrose, sucrose, sorbitol, microcrystalline cellulose, starchand dibasic calcium phosphate dihydrate.

Tablets may also include surface active agents, such as sodium laurylsulfate and polysorbate 80, and glidants such as silicon dioxide andtalc. When present, surface active agents may comprise from about 0.2 wt% to about 5 wt % of the tablet, and glidants may comprise from about0.2 wt % to about 1 wt % of the tablet.

Tablets may also contain lubricants such as magnesium stearate, calciumstearate, zinc stearate, sodium stearyl fumarate, and mixtures ofmagnesium stearate with sodium lauryl sulfate. Lubricants may comprisefrom about 0.25 wt % to about 10 wt % or from about 0.5 wt % to about 3wt % of the tablet.

Tablet blends may be compressed directly or by roller compaction to formtablets. Tablet blends or portions of blends may alternatively be wet-,dry-, or melt-granulated, melt congealed, or extruded before tableting.If desired, prior to blending one or more of the components may be sizedby screening or milling or both. The final dosage form may comprise oneor more layers and may be coated, uncoated, or encapsulated. Exemplarytablets may contain up to about 80 wt % of API, from about 10 wt % toabout 90 wt % of binder, from about 0 wt % to about 85 wt % of diluent,from about 2 wt % to about 10 wt % of disintegrant, and from about 0.25wt % to about 10 wt % of lubricant. For a discussion of blending,granulation, milling, screening, tableting, coating, as well as adescription of alternative techniques for preparing drug products, seeA. R. Gennaro (ed.), Remington: The Science and Practice of Pharmacy(20th ed., 2000); H. A. Lieberman et al. (ed.), Pharmaceutical DosageForms: Tablets, Vol. 1-3 (2d ed., 1990); and D. K. Parikh & C. K.Parikh, Handbook of Pharmaceutical Granulation Technology, Vol. 81(1997).

Consumable oral films for human or veterinary use are pliablewater-soluble or water-swellable thin film dosage forms which may berapidly dissolving or mucoadhesive. In addition to the API, a typicalfilm includes one or more film-forming polymers, binders, humectants,plasticizers, stabilizers or emulsifiers, viscosity-modifying agents,and solvents. Other film ingredients may include anti-oxidants,colorants, flavorants and flavor enhancers, preservatives, salivarystimulating agents, cooling agents, co-solvents (including oils),emollients, bulking agents, anti-foaming agents, surfactants, andtaste-masking agents. Some components of the formulation may performmore than one function.

In addition to dosing requirements, the amount of API in the film maydepend on its solubility. If water soluble, the API would typicallycomprise from about 1 wt % to about 80 wt % of the non-solventcomponents (solutes) in the film or from about 20 wt % to about 50 wt %of the solutes in the film. A less soluble API may comprise a greaterproportion of the composition, typically up to about 88 wt % of thenon-solvent components in the film.

The film-forming polymer may be selected from natural polysaccharides,proteins, or synthetic hydrocolloids and typically comprises from about0.01 wt % to about 99 wt % or from about 30 wt % to about 80 wt % of thefilm.

Film dosage forms are typically prepared by evaporative drying of thinaqueous films coated onto a peelable backing support or paper, which maycarried out in a drying oven or tunnel (e.g., in a combinedcoating-drying apparatus), in lyophilization equipment, or in a vacuumoven.

Useful solid formulations for oral administration may include immediaterelease formulations and modified release formulations. Modified releaseformulations include delayed-, sustained-, pulsed-, controlled-,targeted-, and programmed-release. For a general description of suitablemodified release formulations, see U.S. Pat. No. 6,106,864. For detailsof other useful release technologies, such as high energy dispersionsand osmotic and coated particles, see Verma et al, PharmaceuticalTechnology On-line (2001) 25(2): 1-14.

As noted above, the complex may be combined with one or more otherpharmaceutically active compounds to treat various diseases, disordersor conditions. In such cases, the active compounds may be combined in asingle dosage form as described above or may be provided in the form ofa kit which is suitable for co-administration of the compositions. Thekit comprises (1) two or more different pharmaceutical compositions, atleast one of which contains the complex of the compound of Formula 1 ortautomer thereof; and (2) a device for separately retaining the twopharmaceutical compositions, such as a divided bottle or a divided foilpacket. An example of such a kit is the familiar blister pack used forthe packaging of tablets or capsules. The kit is suitable foradministering different types of dosage forms (e.g., oral andparenteral) or for administering different pharmaceutical compositionsat separate dosing intervals, or for titrating the differentpharmaceutical compositions against one another. To assist with patientcompliance, the kit typically comprises directions for administrationand may be provided with a memory aid.

For administration to human patients, the total daily dose of thecompound of Formula 1, stereoisomer thereof, or tautomer of the compoundof Formula 1 or stereoisomer thereof, is typically in the range of about1 mg to about 3000 mg for oral administration. The total daily dose maybe administered in single or divided doses and, at the physician'sdiscretion, may fall outside of the typical ranges given above. Althoughthese dosages are based on an average human subject having a mass ofabout 60 kg to about 70 kg, the physician will be able to determine theappropriate dose for a patient (e.g., an infant) whose mass fallsoutside of this weight range.

As noted above, the complex may be used to treat diseases, disorders orconditions for which inhibition of BTK is indicated. Such diseases,disorders or conditions generally relate to any unhealthy or abnormalstate in a subject for which the inhibition of BTK provides atherapeutic benefit. More particularly, such diseases, disorders orconditions may involve the immune system and inflammation, includingType I hypersensitivity (allergic) reactions (allergic rhinitis,allergic asthma, and atopic dermatitis); autoimmune diseases (rheumatoidarthritis, multiple sclerosis, systemic lupus erythematosus, psoriasis,lupus nephritis, immune thrombocytopenic purpura, Sjögren's syndrome,ankylosing spondylitis, and Behcet's disease); inflammatory boweldisease; inflammation of the lung (chronic obstructive pulmonarydisease), atherosclerosis, thrombosis, and myocardial infarction. Thecompounds of Formula 1 may also be used to treat diseases, disorders orconditions related to abnormal cell growth, including hematologicalmalignancies, such as acute myeloid leukemia, B-cell chronic lymphocyticleukemia, B-cell lymphoma (e.g., mantle cell lymphoma), T-cell lymphoma(e.g., peripheral T-cell lymphoma), and multiple myeloma, as well asepithelial cancers (i.e., carcinomas), such as lung cancer (small celllung cancer and non-small cell lung cancer), pancreatic cancer, andcolon cancer.

In addition to the hematological malignancies and epithelial cancersnoted above, the complex may also be used to treat other types ofcancer, including leukemia (chronic myelogenous leukemia and chroniclymphocytic leukemia); breast cancer, genitourinary cancer, skin cancer,bone cancer, prostate cancer, and liver cancer; brain cancer; cancer ofthe larynx, gall bladder, rectum, parathyroid, thyroid, adrenal, neuraltissue, bladder, head, neck, stomach, bronchi, and kidneys; basal cellcarcinoma, squamous cell carcinoma, metastatic skin carcinoma,osteosarcoma, Ewing's sarcoma, reticulum cell sarcoma, and Kaposi'ssarcoma; myeloma, giant cell tumor, islet cell tumor, acute and chroniclymphocytic and granulocytic tumors, hairy-cell tumor, adenoma,medullary carcinoma, pheochromocytoma, mucosal neuromas, intestinalganglioneuromas, hyperplastic corneal nerve tumor, marfanoid habitustumor, Wilms' tumor, seminoma, ovarian tumor, leiomyomater tumor,cervical dysplasia, neuroblastoma, retinoblastoma, myelodysplasticsyndrome, rhabdomyosarcoma, astrocytoma, non-Hodgkin's lymphoma,malignant hypercalcemia, polycythermia vera, adenocarcinoma,glioblastoma multiforma, glioma, lymphomas, and malignant melanomas,among others.

In addition to cancer, the complex may also be used to treat otherdiseases, disorders or conditions related to abnormal cell growth,including non-malignant proliferative diseases such as benign prostatichypertrophy, restinosis, hyperplasia, synovial proliferation disorder,idiopathic plasmacytic lymphadenopathy, retinopathy or other neovasculardisorders of the eye, among others.

The complex may also be used to treat autoimmune diseases, disorders orconditions in addition to those listed above. Such diseases, disordersor conditions include Crohn's disease, dermatomyositis, diabetesmellitus type 1, Goodpasture's syndrome, Graves' disease, Guillain-Barrésyndrome, Hashimoto's disease, mixed connective tissue damage,myasthenia gravis, narcolepsy, pemphigus vulgaris, pernicious anemia,polymyositis, primary biliary cirrhosis, temporal arteritis, ulcerativecolitis, vasculitis, and Wegener's granulomatosis, among others.

The complex may be used to treat inflammatory diseases, disorders orconditions including asthma, chronic inflammation, chronic prostatitis,glomerulonephritis, hypersensitivities, inflammatory bowel diseases(ulcerative colitis in addition to Crohn's disease), pelvic inflammatorydisease, reperfusion injury, transplant rejection, vasculitis, andsystemic inflammatory response syndrome.

The complex may also be used to treat specific diseases or conditionsthat may fall within one or more general disorders described above,including arthritis. In addition to rheumatoid arthritis, Sjögren'ssyndrome, systemic lupus erythematosus, SLE in children and adolescents,the complex may also be used to treat other arthritis diseases,including ankylosing spondylitis, avascular necrosis, Behcet's disease,bursitis, calcium pyrophosphate dihydrate crystal deposition disease(pseudo gout), carpal tunnel syndrome, Ehlers-Danlos syndrome,fibromyalgia, Fifth disease, giant cell arteritis, gout, juveniledermatomyositis, juvenile rheumatoid arthritis, juvenilespondyloarthropathy, Lyme disease, Marfan syndrome, myositis,osteoarthritis, osteogenesis imperfect, osteoporosis, Paget's disease,psoriatic arthritis, Raynaud's phenomenon, reactive arthritis, reflexsympathetic dystrophy syndrome, scleroderma, spinal stenosis, Still'sdisease, and tendinitis, among others.

The complex may be combined with one or more pharmacologically activecompounds or therapies for the treatment of one or more diseases,disorders or conditions for which BTK is indicated, including thoseinvolving the immune system, inflammation, and abnormal cell growth. Forexample, the complex may be administered simultaneously, sequentially orseparately in combination with one or more compounds or therapies fortreating arthritis, including rheumatoid arthritis and osteoarthritis,or for treating cancer, including hematological malignancies, such asacute myeloid leukemia, B-cell chronic lymphocytic leukemia, B-celllymphoma, T-cell lymphoma, multiple myeloma, and carcinomas, such aslung cancer, pancreatic cancer, and colon cancer. Such combinations mayoffer significant therapeutic advantages, including fewer side effects,improved ability to treat underserved patient populations, orsynergistic activity.

For example, when used to treat arthritis, the complex may be combinedwith one or more nonsteroidal anti-inflammatory drugs (NSAIDs),analgesics, corticosteroids, biological response modifiers, andprotein-A immunoadsorption therapy. Alternatively or additionally, whentreating rheumatoid arthritis, the complex may be combined with one ormore disease modifying antirheumatic drugs (DMARDs), and when treatingosteoarthritis, the complex may be combined with one or moreosteoporosis agents.

Representative NSAIDs include apazone, aspirin, celecoxib, diclofenac(with and without misoprostol), diflunisal, etodolac, fenoprofen,flurbiprofen, ibuprofen, indomethacin, ketoprofen, meclofenamate sodium,mefenamic acid, meloxicam, nabumetone, naproxen, oxaprozin,phenylbutazone, piroxicam, choline and magnesium salicylates, salsalate,and sulindac. Representative analgesics include acetaminophen andmorphine sulfate, as well as codeine, hydrocodone, oxycodone,propoxyphene, and tramadol, all with or without acetaminophen.Representative corticosteroids include betamethasone, cortisone acetate,dexamethasone, hydrocortisone, methylprednisolone, prednisolone, andprednisone. Representative biological response modifiers include TNF-αinhibitors, such as adalimumab, etanercept, and infliximab; selectiveB-cell inhibitors, such as rituximab; IL-1 inhibitors, such as anakinra,and selective costimulation modulators, such as abatacept.

Representative DMARDs include auranofin (oral gold), azathioprine,chlorambucil, cyclophosamide, cyclosporine, gold sodium thiomalate(injectable gold), hydroxychloroquine, leflunomide, methotrexate,minocycline, mycophenolate mofetil, penicillamine, sulfasalazine, andJAK3 inhibitor (e.g., tofacitinib). Representative osteoporosis agentsinclude bisphosphonates, such as alendronate, ibandronate, risedronate,and zoledronic acid; selective estrogen receptor modulators, such asdroloxifene, lasofoxifene, and raloxifene; hormones, such as calcitonin,estrogens, and parathyroid hormone; and immunosuppressant agents such asazathioprine, cyclosporine, and rapamycin.

Particularly useful combinations for treating rheumatoid arthritisinclude the complex and methotrexate; the complex and one or morebiological response modifiers, such as leflunomide, etanercept,adalimumab, and infliximab; or the complex, methotrexate, and one ormore biological response modifiers, such as leflunomide, etanercept,adalimumab, and infliximab.

For the treatment of thrombus and restenosis, the complex may becombined with one or more cardiovascular agents such as calcium channelblockers, statins, fibrates, beta-blockers, ACE inhibitors, and plateletaggregation inhibitors.

The complex of the compound of Formula 1, stereoisomer thereof, ortautomer of the compound of Formula 1 or stereoisomer thereof, may alsobe combined with one or more compounds or therapies for treating cancer.These include chemotherapeutic agents (i.e., cytotoxic or antineoplasticagents) such as alkylating agents, antibiotics, antimetabolic agents,plant-derived agents, and topoisomerase inhibitors, as well asmolecularly targeted drugs which block the growth and spread of cancerby interfering with specific molecules involved in tumor growth andprogression. Molecularly targeted drugs include both small molecules andbiologics.

Representative alkylating agents include bischloroethylamines (nitrogenmustards, e.g., chlorambucil, cyclophosphamide, ifosfamide,mechlorethamine, melphalan, and uracil mustard); aziridines (e.g.,thiotepa); alkyl alkane sulfonates (e.g., busulfan); nitrosoureas (e.g.,carmustine, lomustine, and streptozocin); nonclassical alkylating agents(e.g., altretamine, dacarbazine, and procarbazine); and platinumcompounds (e.g., carboplatin, cisplatin, nedaplatin, oxaliplatin,satraplatin, and triplatin tetranitrate).

Representative antibiotic agents include anthracyclines (e.g.,aclarubicin, amrubicin, daunorubicin, doxorubicin, epirubicin,idarubicin, pirarubicin, valrubicin, and zorubicin); anthracenediones(e.g., mitoxantrone and pixantrone); and streptomyces (e.g.,actinomycin, bleomycin, dactinomycin, mitomycin C, and plicamycin).

Representative antimetabolic agents include dihydrofolate reductaseinhibitors (e.g., aminopterin, methotrexate, and pemetrexed);thymidylate synthase inhibitors (e.g., raltitrexed and pemetrexed);folinic acid (e.g., leucovorin); adenosine deaminase inhibitors (e.g.,pentostatin); halogenated/ribonucleotide reductase inhibitors (e.g.,cladribine, clofarabine, and fludarabine); thiopurines (e.g.,thioguanine and mercaptopurine); thymidylate synthase inhibitors (e.g.,fluorouracil, capecitabine, tegafur, carmofur, and floxuridine); DNApolymerase inhibitors (e.g., cytarabine); ribonucleotide reductaseinhibitors (e.g., gemcitabine); hypomethylating agent (e.g., azacitidineand decitabine); and ribonucleotide reductase inhibitor (e.g.,hydroxyurea); and an asparagine depleter (e.g., asparaginase)

Representative plant-derived agents include vinca alkaloids (e.g.,vincristine, vinblastine, vindesine, vinzolidine, and vinorelbine),podophyllotoxins (e.g., etoposide and teniposide), and taxanes (e.g.,docetaxel, larotaxel, ortataxel, paclitaxel, and tesetaxel).

Representative type I topoisomerase inhibitors include camptothecins,such as belotecan, irinotecan, rubitecan, and topotecan. Representativetype II topoisomerase inhibitors include amsacrine, etoposide, etoposidephosphate, and teniposide, which are derivatives of epipodophyllotoxins.

Molecularly targeted therapies include biologic agents such as cytokinesand other immune-regulating agents. Useful cytokines includeinterleukin-2 (IL-2, aldesleukin), interleukin 4 (IL-4), interleukin 12(IL-12), and interferon, which includes more than 23 related subtypes.Other cytokines include granulocyte colony stimulating factor (CSF)(e.g., filgrastim) and granulocyte macrophage colony stimulating factor(GM-CSF or CSF2) (e.g., sargramostim, namilumab). Otherimmuno-modulating agents include bacillus Calmette-Guerin, levamisole,and octreotide; monoclonal antibodies against tumor antigens, such astrastuzumab and rituximab; and cancer vaccines, which induce an immuneresponse to tumors.

In addition, molecularly targeted drugs that interfere with specificmolecules involved in tumor growth and progression include inhibitors ofepidermal growth factor (EGF), transforming growth factor-alpha(TGF_(α)), TGF_(β), heregulin, insulin-like growth factor (IGF),fibroblast growth factor (FGF), keratinocyte growth factor (KGF), colonystimulating factor (CSF), erythropoietin (EPO), interleukin-2 (IL-2),nerve growth factor (NGF), platelet-derived growth factor (PDGF),hepatocyte growth factor (HGF), vascular endothelial growth factor(VEGF), angiopoietin, epidermal growth factor receptor (EGFR), humanepidermal growth factor receptor 2 (HER2), HER4, insulin-like growthfactor 1 receptor (IGF1R), IGF2R, fibroblast growth factor 1 receptor(FGF1R), FGF2R, FGF3R, FGF4R, vascular endothelial growth factorreceptor (VEGFR), tyrosine kinase with immunoglobulin-like and epidermalgrowth factor-like domains 2 (Tie-2), platelet-derived growth factorreceptor (PDGFR), Abl, Bcr-Abl, Raf, FMS-like tyrosine kinase 3 (FLT3),c-Kit, Src, protein kinase c (PKC), tropomyosin receptor kinase (Trk),Ret, mammalian target of rapamycin (mTOR), Aurora kinase, polo-likekinase (PLK), mitogen activated protein kinase (MAPK),mesenchymal-epithelial transition factor (c-MET), cyclin-dependentkinase (CDK), Akt, extracellular signal-regulated kinases (ERK), poly(ADP) ribose polymerase (PARP), and the like.

Specific molecularly targeted drugs include selective estrogen receptormodulators, such as tamoxifen, toremifene, fulvestrant, and raloxifene;antiandrogens, such as bicalutamide, nilutamide, megestrol, andflutamide; and aromatase inhibitors, such as exemestane, anastrozole,and letrozole. Other specific molecularly targeted drugs include agentswhich inhibit signal transduction, such as imatinib, dasatinib,nilotinib, trastuzumab, gefitinib, erlotinib, cetuximab, lapatinib,panitumumab, and temsirolimus; agents that induce apoptosis, such asbortezomib; agents that block angiogenesis, such as bevacizumab,sorafenib, and sunitinib; agents that help the immune system destroycancer cells, such as rituximab and alemtuzumab; and monoclonalantibodies which deliver toxic molecules to cancer cells, such asgemtuzumab ozogamicin, tositumomab, 131I-tositumomab, and ibritumomabtiuxetan.

Biological Activity

The activity of compound of Formula 1, stereoisomer thereof, ortautomers of the compound as Formula 1 or stereoisomer thereof, as BTKinhibitors may be determined by a variety of methods, including in vitroand in vivo methods. The following in vitro assay measures a testcompound's ability to inhibit BTK-mediated phosphorylation of aFAM-labeled substrate, 5-FAM-EEPLYWSFPAKKK-NH₂.

Purified BTK may be obtained as follows (Clone SBVC-1603_9P is used). AcDNA sequence encoding residues 382 to 659 of human BTK is cloned intothe vector pSXB4. This construct engineers an in-frame translationalfusion with the Glutathione-S-Transferase (GST) protein for use inaffinity purification. The fusion protein derived from this constructcontains a protease recognition sequence to liberate the BTK from theGST affinity tag. High-titer baculoviral stocks, generated using theBac-to-Bac® system (Invitrogen), are used to express the recombinantprotein in Spodoptera frugiperda Sf9 cells in 10 L Wave bags.Recombinant proteins are isolated from cellular extracts by passage overGlutathione Sepharose 4B (GE Healthcare) and the BTK moiety is releasedfrom the GST affinity tag by treatment with PreScission protease. TheBTK recombinant protein is further purified by size exclusionchromatography (HiLoad 16/60 Superdex 200, GE Healthcare) in a buffercontaining 20 mM Hepes (pH 7.4), 50 mM NaCl, 10 mM MgCl₂, 0.25 mM TCEPand 0.1 mM EDTA. The purity of the fractions is assessed by SDS PAGE andthe peak protein fractions are pooled and concentrated using AmiconUltra-15 Centrifugal Filter Devices (Millipore).

The inhibitory properties of compounds relative to BTK is determinedusing a black 384-well-plate format in a buffer which contains 50 mMHepes, 10 mM NaCl, 10 mM MgCl₂, 0.2 mM EDTA, 0.01% Brij35®, 1 mM DTT,and 0.1 mg/mL BSA at pH 7.3. The test compound is prepared in DMSO using2-fold serial dilutions for 11 data points, which are added to thebuffer so that each dilution contains 3% DMSO. To initiate the assay, 5μL of 3 μM 5FAM-EEPLYWSFPAKKK-NH₂ (in buffer), 5 μL of diluted testcompound (3% DMSO in buffer), and 5 μL of 9 nM BTK and 150 μM ATP inbuffer are combined in each well. The reaction mixtures are incubated atroom temperature for 60 minutes and then quenched by adding 25 μL of 50mM EDTA. To quantify the fluorescent-labeled substrate and productfollowing reaction, the test plate is loaded on a Caliper LC-3000, whichmeasures percent of conversion by microfluidic-based separation.Corresponding IC₅₀ values are calculated by non-linear curve fitting ofthe compound concentrations and percent of inhibition to the standardIC₅₀ equation and reported as pIC₅₀, i.e., −log(IC₅₀), where IC₅₀ ismolar concentration at 50% inhibition.

EXAMPLES

The following examples are intended to be illustrative and non-limiting,and represent specific embodiments of the present invention.

¹H Nuclear magnetic resonance (NMR) spectra were obtained for many ofthe compounds in the following examples. Characteristic chemical shifts(6) are given in parts-per-million downfield from tetramethylsilaneusing conventional abbreviations for designation of major peaks,including s (singlet), d (doublet), t (triplet), q (quartet), m(multiplet), and br (broad). The following abbreviations are used forcommon solvents: CDCl₃ (deuterochloroform), DMSO-d₆(deuterodimethylsulfoxide), CD₃OD (deuteromethanol), CD₃CN(deuteroacetonitrile), and THF-d₈ (deuterotetrahydrofuran). The massspectra (M+H) were recorded using either electrospray ionization(ESI-MS) or atmospheric pressure chemical ionization (APCI-MS).

Where indicated, products of certain preparations and examples arepurified by mass-triggered HPLC (Pump: Waters™ 2525; MS: ZQ™; Software:MassLynx™), flash chromatography or preparative thin layerchromatography (TLC). Reverse phase chromatography is typically carriedout on a column (e.g., Gemini™ 5 μm C18 110 Å, Axia™, 30×75 mm, 5 μm)under acidic conditions (“acid mode”) eluting with ACN and water mobilephases containing 0.035% and 0.05% trifluoroacetic acid (TFA),respectively, or under basic conditions (“basic mode”) eluting withwater and 20/80 (v/v) water/acetonitrile mobile phases, both containing10 mM NH₄HCO₃. Preparative TLC is typically carried out on silica gel 60F₂₅₄ plates. After isolation by chromatography, the solvent is removedand the product is obtained by drying in a centrifugal evaporator (e.g.,GeneVac™), rotary evaporator, evacuated flask, etc. Reactions in aninert (e.g., nitrogen) or reactive (e.g., H₂) atmosphere are typicallycarried out at a pressure of about 1 atmosphere (14.7 psi).

Example 1:(S)-3-(1-((1-acryloylpyrrolidin-3-yl)oxy)isoquinolin-3-yl)-1H-1,2,4-triazol-5(4H)-one

Step A: (S)-tert-butyl3-((3-chloroisoquinolin-1-yl)oxy)pyrrolidine-1-carboxylate

To (S)-tert-butyl 3-hydroxypyrrolidine-1-carboxylate (1.134 g, 6.06mmol) in NMP (10 mL) at 0° C. was added NaH (60%) (202 mg, 5.05 mmol).The mixture was stirred for 5 minutes and 1,3-dichloroisoquinoline(1.000 g, 5.05 mmol) was added. The reaction mixture was stirred at RTfor 5 minutes and then heated at 135° C. for 30 minutes in a microwavereactor. The mixture was diluted with water (400 mL) and extracted withEtOAc (3×125 mL). The organic layers were combined, washed with brine,dried over Na₂SO₄, filtered, and concentrated in vacuo. The crudeproduct was purified by silica column chromatography eluting with agradient of 25-50% EtOAc in hexane to give the title compound (5.29 g,75%). ¹H NMR (500 MHz, DMSO-d₆) δ ppm 1.40 (d, J=14.16 Hz, 9H),2.12-2.34 (m, 2H), 3.42-3.58 (m, 3H), 3.69 (td, J=12.33, 4.64 Hz, 1H),5.63-5.76 (m, 1H), 7.59 (s, 1H), 7.64 (ddd, J=8.30, 7.08, 1.22 Hz, 1H),7.81 (td, J=7.57, 1.46 Hz, 1H), 7.87-7.92 (m, 1H), 8.11-8.19 (m, 1H);ESI-MS m/z [M+H-tert-butyl]⁺ 293.5.

Step B: (S)-tert-butyl3-((3-cyanoisoquinolin-1-yl)oxy)pyrrolidine-1-carboxylate

A solution of (S)-tert-butyl3-((3-chloroisoquinolin-1-yl)oxy)pyrrolidine-1-carboxylate (4.430 g,12.70 mmol), zinc cyanide (2.980 g, 25.40 mmol) and Pd(PPh₃)₄ (1.468 g,1.27 mmol) in DMF (36.3 mL) was heated at 160° C. for 20 minutes in amicrowave reactor. The reaction mixture was filtered, diluted with water(400 mL) and extracted with EtOAc (2×100 mL). The organic layers werecombined, washed with brine, dried over Na₂SO₄, and concentrated invacuo. The crude product was purified by silica column chromatography togive the title compound as a white-to-pale-yellow solid (3.570 g, 83%).¹H NMR (500 MHz, DMSO-d₆) δ ppm 1.40 (d, J=13.18 Hz, 9H), 2.23 (d,J=11.23 Hz, 2H), 3.42-3.59 (m, 3H), 3.65-3.75 (m, 1H), 5.68-5.80 (m,1H), 7.82-7.89 (m, 1H), 7.91-7.98 (m, 1H), 8.06 (d, J=8.79 Hz, 1H),8.21-8.30 (m, 2H); ESI-MS m/z [M+H-tert-butyl]⁺ 284.6.

Step C: (S)-tert-butyl3-((3-(5-oxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)isoquinolin-1-yl)oxy)pyrrolidine-1-carboxylate

(S)-tert-Butyl 3-((3-cyanoisoquinolin-1-yl)oxy)pyrrolidine-1-carboxylate(4.670 g, 13.76 mmol), ethyl hydrazinecarboxylate (7.160 g, 68.80 mmol),DBU (1.037 mL, 6.88 mmol) and NMP (34.6 mL) were mixed in a 200 mL highpressure reaction vessel. The resulting suspension was heated at 170° C.overnight and was then cooled to room temperature. Crushed ice was addedand the mixture was stirred. A yellow precipitate was collected byvacuum filtration, washed with additional water, and dried in a vacuumoven at 45° C. overnight to give the title compound, which was used inthe next step without further purification (5.47 g). ¹H NMR (500 MHz,DMSO-d₆) δ ppm 1.33-1.51 (m, 9H), 2.09-2.38 (m, 2H), 3.39-3.60 (m, 3H),3.75 (dd, J=12.20, 4.88 Hz, 1H), 6.03-6.22 (m, 1H), 7.62-7.71 (m, 1H),7.81 (td, J=7.57, 1.46 Hz, 1H), 7.95-8.05 (m, 1H), 8.11-8.29 (m, 2H),11.78 (s, 1H), 12.03 (br s, 1H).

Step D:(S)-3-(1-(pyrrolidin-3-yloxy)isoquinolin-3-yl)-1H-1,2,4-triazol-5(4H)-one

To a 200 mL round-bottom flask charged with crude (S)-tert-butyl3-((3-(5-oxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)isoquinolin-1-yl)oxy)pyrrolidine-1-carboxylate(5.47 g) and dioxane (27.5 mL) was added 4M HCl in dioxane (13.76 mL,55.1 mmol). The suspension was stirred at RT with periodic monitoring byHPLC. Upon completion, the reaction mixture was concentrated in vacuo togive an HCl salt of the title compound as a light tan powder that wasdried and used without further purification. ESI-MS m/z [M+H]⁺ 298.6.

Step E:(S)-3-(1-((1-acryloylpyrrolidin-3-yl)oxy)isoquinolin-3-yl)-1H-1,2,4-triazol-5(4H)-one

To a suspension of(S)-3-(1-(pyrrolidin-3-yloxy)isoquinolin-3-yl)-1H-1,2,4-triazol-5(4H)-onehydrochloride (4.29 g) in DCM (48.1 mL) was added 2,6-dimethylpyridine(3.19 mL, 27.4 mmol). Upon cooling the suspension to 0° C., acryloylchloride (1.3 mL, 15.9 mmol) was added drop-wise. The reaction mixturewas stirred for 15 minutes and warmed to RT over a period of 90 minutes.Additional 2,6-dimethylpyridine (1.68 mL, 14.43 mmol) and acryloylchloride (0.469 mL, 5.77 mmol) were added and the mixture was stirreduntil HPLC indicated the reaction was completed. The product wascollected by vacuum filtration, washed with DCM, and dried to give titlecompound as a pale yellow solid (1.929 g, 39.9% over 3 steps). ¹H NMR(500 MHz, DMSO-d) 6 ppm 2.16-2.43 (m, 2H), 3.58-3.73 (m, 1H), 3.74-3.91(m, 2H), 4.10 (dd, J=11.72, 4.88 Hz, 1H), 5.60-5.74 (m, 1H), 6.10-6.25(m, 2H), 6.53-6.73 (m, 1H), 7.62-7.69 (m, 1H), 7.77-7.85 (m, 1H),7.95-8.05 (m, 2H), 8.17 (d, J=8.30 Hz, 1H), 11.78 (s, 1H), 12.03 (d,J=13.18 Hz, 1H); ESI-MS m/z [M+H]⁺ 352.6.

Example 2:(R)-3-(1-((1-acryloylpyrrolidin-3-yl)oxy)isoquinolin-3-yl)-1H-1,2,4-triazol-5(4H)-one

Step A: (R)-tert-butyl3-((3-cyanoisoquinolin-1-yl)oxy)pyrrolidine-1-carboxylate

A mixture of (R)-tert-butyl 3-hydroxypyrrolidine-1-carboxylate (496 mg,2.65 mmol) in NMP (4 mL) at 0° C. was treated with NaH (106 mg, 2.65mmol) and stirred for 1 hour. Next, 1-chloroisoquinoline-β-carbonitrile(500 mg, 2.65 mmol) was added and the reaction mixture was stirred at RTfor 15 minutes and then heated at 140° C. for 15 minutes in a microwavereactor. The crude reaction mixture, which contained the title compound,was used directly in the next step.

Step B: (R)-tert-butyl3-((3-(5-oxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)isoquinolin-1-yl)oxy)pyrrolidine-1-carboxylate

To crude (R)-tert-butyl3-((3-cyanoisoquinolin-1-yl)oxy)pyrrolidine-1-carboxylate was addedethyl hydrazinecarboxylate (1.104 g, 10.60 mmol). The reaction mixturewas heated at 175° C. overnight and was subsequently cooled and dilutedwith EtOAc. The organic phase was washed with brine, dried over Na₂SO₄,and concentrated to give the title compound, which was used directly inthe next step.

Step C:(R)-3-(1-(pyrrolidin-3-yloxy)isoquinolin-3-yl)-1H-1,2,4-triazol-5(4H)-one

To crude (R)-tert-butyl3-((3-(5-oxo-4,5-dihydro-1H-1,2,4-triazol-3-yl)isoquinolin-1-yl)oxy)pyrrolidine-1-carboxylatewas added a minimal amount of NMP and TFA (2 mL). The solution wasstirred at RT for 10 minutes and concentrated. The crude product waspurified by preparative HPLC eluting with a gradient of 15-22% ACN inwater (acid mode) to give the title compound (229 mg, 29% over 3 steps).

Step D:(R)-3-(1-((1-acryloylpyrrolidin-3-yl)oxy)isoquinolin-3-yl)-1H-1,2,4-triazol-5(4H)-one

To a solution of(R)-3-(1-(pyrrolidin-3-yloxy)isoquinolin-3-yl)-1H-1,2,4-triazol-5(4H)-one(11 mg, 0.037 mmol) in DCM (3 mL) was added 2,6-dimethylpyridine (5.80μL, 0.050 mmol) at 0° C. followed by acryloyl chloride (8.08 μL, 0.100mmol). The reaction mixture was stirred at RT overnight, forming a whitesolid. The solids were filtered and dried to give the title compound (4mg, 23%). ¹H NMR (400 MHz, CD₃CN) 6 ppm 2.22-2.51 (m, 2H), 3.09-3.17 (m,1H), 3.68-3.91 (m, 2H), 3.94 (br s, 1H), 4.06 (d, J=12.13 Hz, 1H),5.60-5.76 (m, 1H), 5.98 (br s, 1H), 6.06 (br s, 1H), 7.47-7.62 (m, 1H),7.62-7.74 (m, 1H), 7.79 (d, J=7.58 Hz, 1H), 7.92 (d, J=3.79 Hz, 1H),8.15 (d, J=8.08 Hz, 1H); ESI-MS m/z [M+H]⁺ 352.0.

TABLE 2, below, lists BTK inhibition data for many of the compoundsdescribed in the examples, where larger pIC₅₀ values represent higherpotency. The compounds were tested in accordance with the assaydescribed in the specification.

TABLE 2 BTK Inhibition (pIC₅₀) for Example Compounds Example No. pIC₅₀1 >8.7 2 7.1

Example 3: Pharmacokinetics Study in Dogs

A pharmacokinetic analysis was conducted in male beagles following oraladministration of the compound of Example 1 complexed with CAPTISOL®(Formulations A and E) or formulated in various solid dosage forms(Formulations B, C, and F). Formulation D is a capsule containing aco-crystal of the compound of Example 1. In the formulations, below, theamounts of the compound of Example 1 listed in Tables 3-7 correspond tothe desired amount of the compound in the respective formulation (i.e.,without impurities).

A. Formulation a (Aqueous CAPTISOL® Solution)

Table 3 lists the components of Formulation A. After dissolving thecompound of Example 1 in 0.25M aqueous NaOH solution, a 40% w/v aqueousCAPTISOL® solution was added to obtain a 1:25 (w/w) activecompound:CAPTISOL® ratio. The pH was adjusted to 7 using 10% phosphoricacid, resulting in an aqueous solution having an active compoundconcentration of approximately 7.5 mg/mL. The dosage was set at 100 mgand approximately 13.3 mL was administered orally to each test subject.

TABLE 3 Composition of Formulation A Component Amount Compound ofExample 1 2.25¹ g 0.25M NaOH (aq) 36 mL 40% (w/v) CAPTISOL ® 150 mL 10%(w/w) phosphoric acid (aq) 4.92 mL Water q.s. Total 300 mL ¹Amount ofExample 1 compound without impurities

Formulation B (Immediate Release Tablet)

Table 4 lists the components of Formulation B. Water was added to a drymixture of the compound of Example 1, binders and disintegrants. The wetmixture was granulated using a mortar and pestle and then dried toobtain a granulated powder. A lubricant was admixed with the granulatedpowder and a tabletop tablet molding machine (HANDTAB-200, IchihashiSeiki Co.) was used to prepare tablets from the resulting powdermixture. Each tablet contained 300 mg of the compound of Example 1 andwas formed under a tableting force of 10 kN (total weight ˜400 mg, 12 mmlong diameter×7 mm short diameter). The dosage was set at 300 mg and onetablet was administered orally to each test subject.

TABLE 4 Composition of Formulation B Component Amount/Tablet Compound¹of Example 1 300 mg Binder² 43 mg Binder/Disintegrant 30 mgBinder/Disintegrant 9 mg Binder/Disintegrant 15 mg Lubricant 3 mg Water³q.s. Total 400 mL ¹Amount of Example 1 compound without impurities²Nominal amount of binder; actual amount accounts for impurities in theAPI ³Water was removed during processing

Formulation C (Physical Mixture of Example 1 Compound and CAPTISOL®):

Table 5 lists the components of Formula C. A 1:25 (w/w) ratio of thecompound of Example 1 and CAPTISOL®, microcrystalline cellulose, sodiumcarboxymethyl starch, and magnesium stearate were mixed using a mortarand pestle. A tabletop tablet molding machine (HANDTAB-200, IchihashiSeiki Co.) was used to prepare tablets from the resulting powdermixture. Each tablet contained 25 mg of the compound of Example 1 andwas formed under a tableting force of 12 kN (total weight ˜700 mg, 18.5mm long diameter×9 mm short diameter). The dosage was set at 100 mg andfour tablets were administered orally to each test subject.

TABLE 5 Composition of Formulation C Component Amount/Tablet Compound¹of Example 1 25 mg CAPTISOL ® 625 mg Microcrystalline cellulose² 15 mgSodium carboxymethyl starch 30 mg Magnesium stearate 5 mg Total 700 mL¹Amount of Example 1 compound without impurities ²Nominal amount; actualamount accounts for impurities in the API

Formulation D (Co-Crystal Capsule)

A gelatin capsule containing a co-crystal of the compound of Example 1was prepared. Each capsule contained 100 mg of the compound of Example 1(144 mg of co-crystal). One capsule was administered orally to each testsubject.

Formulation E (CAPTISOL® Spray-Dried Dispersion Tablet)

Table 6 lists the components of Formula E. Aqueous Formulation A, above,was spray dried using a PSD-1 spray drier (GEA Niro) to obtain a solidcomplex of the compound of Example 1 and CAPTISOL®. The complex wasadmixed with light anhydrous silicic acid and magnesium stearate using amortar and pestle. A tabletop tablet molding machine (HANDTAB-200,Ichihashi Seiki Co.) was used to prepare tablets from the resultingpowder mixture. Each tablet contained 25 mg of the compound of Example 1and was formed under a tableting force of 12 kN (total weight ˜711 mg,18.5 mm long diameter×9 mm short diameter). The dosage was set at 100 mgand four tablets were administered orally to each test subject.

TABLE 6 Composition of Formulation E Component Amount/Tablet Spray driedCompound¹ of Example 1 25 mg powder CAPTISOL ® 667 mg NaOH 4 mgphosphoric acid 5.47 mg Excipients Light anhydrous silicic acid 5 mgMagnesium stearate 5 mg Total 711.47 mg ¹Amount of Example 1 compoundwithout impurities in spray dried powder

Formulation F (Solid Dispersion Tablet)

Table 7 lists the components of Formula F. The compound of Example 1 andhypromellose phthalate were dissolved at a ratio of 1:40 (w/w) indimethyl sulfoxide. The resulting solution was lyophilized using afreeze dryer (FDU-2100, EYELA) to obtain a solid dispersion powder,which was granulated using a mortar and pestle. The granulated powderwas admixed with D-mannitol, microcrystalline cellulose, croscarmellosesodium, light anhydrous silicic acid, and magnesium stearate. A tabletoptablet molding machine (HANDTAB-200, Ichihashi Seiki Co.) was used toprepare tablets from the resulting powder mixture. Each tablet contained25 mg of the compound of Example 1 and was formed under a tabletingforce of 15 kN (tablet total weight ˜600 mg, 16 mm long diameter×9 mmshort diameter). The dosage was set at 100 mg and four tablets wereadministered orally to each test subject.

TABLE 7 Composition of Formulation F Component Amount/Tablet Compound¹of Example 1 25 mg Hypromellose phthalate 100 mg D-mannitol 360 mgMicrocrystalline cellulose 60 mg Croscarmellose sodium 40 mg Lightanhydrous silicic acid 9 mg Magnesium stearate 6 mg Total 600 mL ¹Amountof Example 1 compound without impurities

Each formulation described above was administered orally to a fastingbeagle (one-year old male, five test subjects per formulation). Apentagastrin solution was administered to each test subject 15 minutesprior to the administration of drug. Blood samples were taken at 15 and30 minutes, and at 1, 2, 4, 6, 8, 12, and 24 hours after administrationof drug. Plasma was obtained using centrifugation. The concentration ofExample 1 compound in plasma was measured using LC and MS/MS under theconditions listed in Tables 8 and 9, respectively.

TABLE 8 LC conditions Analytical column Kinetex C18, 50 mm × 2.0 mmI.D., 2.6 μm (Phenomenex) Column oven temperature 40° C. Mobile phasePurified water/acetonitrile/formic acid (600:200:0.1, v/v/v) Flow rate0.2 mL/minute Injection volume 20 μL Auto sampler temperature 10° C.Rinsing solution Acetonitrile/purified water/formic acid (600:400:0.1,v/v/v) Run time 5.0 minutesThe Effluent from 2.0 to 5.0 minutes was transferred to the MS/MS byvalve operation.

TABLE 9 MS/MS conditions Ionization mode Turbo ion spray PolarityPositive Scan type Selected reaction monitoring (SRM) Ion spray voltage(IS) 5500 V Turbo probe temp. (TEM) 600° C. Interface heater (ihe) ONCurtain gas pressure (CUR) 0.28 MPa (40 psi, N₂) Ion source gas 1pressure (GS1) 0.28 MPa (40 psi, Air) Ion source gas 2 pressure (GS2)0.28 MPa (40 psi, Air) Collision gas pressure (CAD) 8 Bit (N₂) Dwelltime 0.8 seconds (for compound of Example 1) 0.2 seconds (for internalstandard) Duration time 5.0 minutes Monitor ion Precursor Product DP¹EP² CE³ CXP⁴ and parameters ion (m/z) → ion (m/z) (V) (V) (V) (V)Compound of Example 1 352.3 → 124.1 131 10 23 20 Internal Standard 355.0→ 127.2 106 10 23 18 ¹Declustering Potential, ²Entrance Potential,³Collision Energy, ⁴Collision Cell Exit Potential

FIG. 1 shows the mean concentration of the compound of Example 1 inblood plasma as a function of time following oral dosing of dogs withFormulations A, B, C, D, E, and F. The maximum concentration of theactive compound in blood (C_(max)) and the time to reach the maximumconcentration of the drug in blood (t_(max)) were measured using theconcentration vs. time curves. The concentration of the drug in bloodfrom 0 to 24 hours and the area under the concentration-versus-timecurve (AUC_(0-24 h)) were calculated using a linear trapezoidal method.Table 10 lists pharmacokinetic data (average values (S.D.)).

TABLE 10 Dog Pharmacokinetics Formu- Dose t_(max) C_(max) AUC_(0-24 h)lation (mg/subject) (h) (ng/mL) (ng · h/mL) A 100 0.8 (0.3) 1251.4(717.2) 2353.0 (864.9) B 300 1.6 (0.5) 4.7 (1.0) 30.2 (14.5) C 100 0.6(0.2) 9.3 (1.3) 36.9 (1.7) D 100 1.3 (0.7) 229.0 (106.5) 585.5 (231.6) E100 0.9 (0.3) 1133.3 (679.5) 1994.6 (798.7) F 100 0.4 (0.1) 51.5 (29.8)130.2 (88.3)

Example 4: Dissolution Test

FIG. 2 shows the mean eluent concentration of the compound of Example 1as a function of time for Formulations A, B, C, and E, which wereevaluated in the Japanese Pharmacopoeia Dissolution Test using thepaddle method. The test was performed in 900 mL of the JapanesePharmacopoeia Test Fluid No. 2 for the dissolution test (pH=6.8) usingan NTR 6200AT dissolution testing apparatus (Toyama Co., Ltd.) with apaddle rotation speed of 100 rpm. For each formulation, test sampleswere collected at prescribed time points, filtered through a 0.45 μmpolyproplylene membrane filter (GHP Acrodisc Glass Fiber Pre-filter,PALL), and diluted in dimethyl sulfoxide. The eluent concentration ofthe compound of Example 1 was measured using UPLC under conditionsdescribed in Table 11.

TABLE 11 UPLC conditions Analytical column Waters Acquity BEH C18, 100mm × 2.1 mm, 1.7 μm Column temperature 50° C. Detection UV @ 250 nmMobile phase 0.025% TFA in Water/0.02% TFA in acetonitrile (7/3, v/v)Flow rate 0.3 mL/minute Injection volume 1 μL Auto sampler 25° C.temperature Rinsing solution Acetonitrile/purified water (1/1, v/v) Runtime 4.0 minutes

As used in this specification and the appended claims, singular articlessuch as “a,” “an,” and “the,” may refer to a single object or to aplurality of objects unless the context clearly indicates otherwise.Thus, for example, reference to a composition containing “a compound”may include a single compound or two or more compounds. It is to beunderstood that the above description is intended to be illustrative andnot restrictive. Many embodiments will be apparent to those of skill inthe art upon reading the above description. Therefore, the scope of theinvention should be determined with reference to the appended claims andincludes the full scope of equivalents to which such claims areentitled. The disclosures of all articles and references, includingpatents, patent applications and publications, are herein incorporatedby reference in their entirety and for all purposes.

1. A complex comprising a compound of Formula 1,

a stereoisomer thereof or a tautomer of the compound of Formula 1 orstereoisomer thereof, and a cyclodextrin, wherein the complex is anamorphous solid.
 2. The complex according to claim 1, wherein thecompound is(S)-3-(1-((1-acryloylpyrrolidin-3-yl)oxy)isoquinolin-3-yl)-1H-1,2,4-triazol-5(4H)-oneor a tautomer thereof.
 3. The complex according to claim 1, wherein thecompound is(R)-3-(1-((1-acryloylpyrrolidin-3-yl)oxy)isoquinolin-3-yl)-1H-1,2,4-triazol-5(4H)-oneor a tautomer thereof.
 4. The complex according to claim 1, wherein thecyclodextrin is an unmodified β-cyclodextrin or a β-cyclodextrinderivative.
 5. The complex according to claim 1, wherein thecyclodextrin is a cyclodextrin derivative.
 6. The complex according toclaim 1, wherein the cyclodextrin is selected from unmodifiedα-cyclodextrin, unmodified β-cyclodextrin, unmodified γ-cyclodextrin,methyl-β-cyclodextrin, dimethyl-β-cyclodextrin,trimethyl-β-cyclodextrin, randomly methylated-β-cyclodextrin, randomlydimethylated-β-cyclodextrin, ethyl-β-cyclodextrin,diethyl-β-cyclodextrin, triethyl-β-cyclodextrin,(2-hydroxyethyl)-β-cyclodextrin, (2-hydroxypropyl)-β-cyclodextrin,(2-hydroxypropyl)-γ-cyclodextrin, (β-hydroxypropyl)-β-cyclodextrin,(2,3-dihydroxypropyl)-β-cyclodextrin,(2-hydroxyisobutyl)-β-cyclodextrin, carboxymethyl-β-cyclodextrin,carboxymethyl ethyl-β-cyclodextrin, tributyryl-β-cyclodextrin,trivaleryl-β-cyclodextrin, dihexanoyl-β-cyclodextrin, sulfobutyletherβ-cyclodextrin, glucosyl-β-cyclodextrin, and maltosyl-β-cyclodextrin. 7.The complex according to claim 1, wherein the cyclodextrin is selectedfrom unmodified β-cyclodextrin, methyl-β-cyclodextrin,(2-hydroxypropyl)-β-cyclodextrin, (2-hydroxypropyl)-γ-cyclodextrin, andsulfobutylether β-cyclodextrin.
 8. The complex according to claim 1,wherein the cyclodextrin is a sulfobutylether-β-cyclodextrin or a(2-hydroxypropyl)-β-cyclodextrin.
 9. The complex according to claim 1,wherein the cyclodextrin is a sulfobutylether-β-cyclodextrin.
 10. Thecomplex according to claim 1, wherein the cyclodextrin and the compoundof Formula 1, stereoisomer or tautomer are present in a molar ratio ofabout 1:1 to about 10:1.
 11. The complex according to claim 1, whereinthe cyclodextrin and the compound of Formula 1, stereoisomer or tautomerare present in a molar ratio of about 1:1 to about 5:1.
 12. The complexaccording to claim 1, wherein the cyclodextrin and the compound ofFormula 1, stereoisomer or tautomer are present in a molar ratio ofabout 1:1.
 13. A pharmaceutical composition comprising a complex asdefined in claim 1; and a pharmaceutically acceptable excipient.
 14. Amethod of making a complex comprising the compound of Formula 1,

a stereoisomer thereof or a tautomer of the compound of Formula 1 orstereoisomer thereof, and a cyclodextrin, wherein the complex is anamorphous solid, the method comprising: atomizing a liquid solution intodroplets, the liquid solution comprising the compound, stereoisomer, ortautomer of Formula 1, a cyclodextrin derivative, and water; andremoving at least a portion of the water from the droplets to form thecomplex.
 15. The method according to claim 14, wherein the liquidsolution was obtained by dissolving the compound, stereoisomer ortautomer in water having a pH of about 12 or greater.
 16. The methodaccording to claim 15, wherein the liquid solution was obtained bydissolving the compound, stereoisomer or tautomer in water having a pHof about 13 or greater.
 17. The method according to claim 14, whereinthe pH of the liquid solution was adjusted to a pH of about 7 beforeatomizing the liquid solution into droplets.
 18. A complex as defined inclaim 1 for use as a medicament.
 19. A method for inhibiting BTK in asubject, the method comprising administering to the subject a complex asdefined in claim
 1. 20. A method of treating a disease, disorder orcondition in a subject, the method comprising administering to thesubject an effective amount of a complex as defined in claim 1, whereinthe disease, disorder or condition is associated with BTK.
 21. A Themethod of treating a disease, disorder or condition in a subject, themethod comprising administering to the subject an effective amount of acomplex as defined in claim 1, wherein the disease, disorder orcondition is selected from Type I hypersensitivity reactions, autoimmunediseases, inflammatory disorders, cancer, and non-malignantproliferative disorders.
 22. A method of treating a disease, disorder orcondition in a subject, the method comprising administering to thesubject an effective amount of a complex as defined in claim 1, whereinthe disease, disorder or condition is selected from allergic rhinitis,asthma, atopic dermatitis, rheumatoid arthritis, multiple sclerosis,systemic lupus erythematosus, lupus nephritis, psoriasis, immunethrombocytopenic purpura, inflammatory bowel disease, chronicobstructive pulmonary disease, Sjögren's syndrome, ankylosingspondylitis, Behcet's disease, graft versus host disease, pemphigusvulgaris, idiopathic plasmacytic lymphadenopathy, atherosclerosis,myocardial infarction, and thrombosis.
 23. A method of treating adisease, disorder or condition in a subject, the method comprisingadministering to the subject an effective amount of a complex as definedin claim 1, wherein the disease, disorder or condition is selected fromB-cell lymphoma, chronic lymphocytic leukemia, and multiple myeloma. 24.A combination comprising an effective amount of a complex as defined inclaim 1, and at least one additional pharmacologically active agent. 25.The combination according to claim 24, wherein the additionalpharmacologically active agent is a disease modifying antirheumatic drug(DMARD).
 26. The combination according to claim 25, wherein the DMARD ismethotrexate.